[Purposes]To investigate the relationship between environmental DNA sequence abundance and species number in a pond model. [Methods]In September 2022, Myxocyprinus asiaticus, Leiocassis longirostris, Megalobrama pellegrini and Spinibarbus sinensis were temporarily cultured in a specific size pond. After 10 days, 15 sampling sites were set up in this pond. The sequences abundance of the above four fish species were detected through a series of standardized analytical processes, including water sample collection, filtration, eDNA extraction, PCR amplification, high-throughput sequencing and biosignature analysis using the mitochondrial gene 12S gene as a marker . The PCR amplification results, rarefaction curve, species number and sequence abundance trend were analyzed with the use of Excel software, Prism software and R software package. [Findings]Under the pond model, the sequence abundance of M. asiaticus was the lowest (3970 sequences), and that of S. sinensis was the highest (687,895 sequences). The sequence abundance of the four fish species showed a positive trend with the number of species. [Conclusions]In the pond model, there was a positive trend between the sequence abundance and species number of M. asiaticus, L. longirostris, M. pellegrini and S. sinensis, which could provide a certain scientific basis for the subsequent application of eDNA technology to the assessment of relative abundance of fishes in natural river systems.